When flu viruses join forces

Certain influenza variants infect cells better together than apart, new study shows
Viruses in shape of fish attacking a cell
A new study has revealed that different variants of the flu virus cooperate to infect cells, further evidence of the complex viral communities that underlie infections. Illustration by Jacqueline Morgado / Fred Hutch News Service

From humans to bats to honeybees, working together to get ahead is inherent. Cooperation is part of who we are as social creatures.

But it turns out entities as simple as the flu virus — housing a mere eight genes inside a microscopic protein shell and debatably not even alive — can cooperate too. Discoveries of viruses working together and the importance of genetic diversity among viruses may even shift how we think about infection, researchers say.

“Once a virus initiates an infection and starts to replicate, it quickly generates a lot of mutant viruses,” said Fred Hutchinson Cancer Research Center evolutionary biologist Dr. Jesse Bloom of the complex community that can arise from infection. “It’s not right to think that all the viruses in an infection are identical.”

Bloom and his colleagues from Fred Hutch and the University of Washington have found that two variants (the original and a mutated version) of influenza H3N2, a virus responsible for many cases of the yearly seasonal flu, can join forces — in a laboratory setting, at least.

The two strains, which differ at only a single point in their genomes, infect cells in a petri dish more efficiently together than alone.

Dr. Jesse Bloom
"It’s not right to think that all the viruses in an infection are identical,” said Dr. Jesse Bloom. The evolutionary biologist led a study that found that flu viruses produce mutations that join forces to infect cells and grow better together than alone. Photo by Robert Hood / Fred Hutch News Service

The researchers, led by Bloom and UW graduate student Katherine Xue, published their findings in the journal eLife on Tuesday. They haven’t yet uncovered whether this cooperation plays a role in human infections as well as in the lab — they’re currently looking at samples taken from people with the flu to try to answer that question.

Researchers have previously shown that other viruses, such as polio and measles, may also cooperate by relying on a mix of genetically diverse strains to efficiently infect cells or animals. It’s also known that each person infected with a flu strain becomes home to a huge number of genetically diverse viruses, but researchers don’t yet understand whether that variety translates to true cooperation or just reflects flu’s rapid evolution.

“The crucial discovery is not that this mutation occurs, because that’s already been reported,” Bloom said. “The crucial discovery is that a mutation can help two different viruses grow better together than they do on their own. We think the principle could potentially extend well beyond this specific mutation.”

Annoying lab artifact or viral evolution in action?

The Hutch researchers were far from the first to notice this particular variation in flu; it’s been reported hundreds of times before, they said. Flu surveillance groups, primarily labs working with the World Health Organization, had noticed that the two viral variants frequently appear together when strains from flu-infected people are grown in the lab. But researchers assumed this was just an adaptation to life under artificial conditions, Xue said. In fact, the previous publications describing this variant were often framed in the context of how to get rid of it — the mutation can interfere with some lab assays.

WHO labs study patient samples to understand which flu strains are circulating in the world to inform yearly vaccine design. Their standard practice is to “passage” each patient sample in cells in the lab, letting the virus infect cells in a petri dish and then adding newly produced viruses from that dish to another collection of cells, and so on. Only after the virus is passaged do the researchers determine its genetic sequence (or sequences). So the research groups assumed the mix of flu variants was cropping up in the lab passaging, not in humans.

Something about that assumption didn’t add up for Xue and Bloom. For one, if the variant was truly a result of the virus adapting to petri dish life, you’d expect it to quickly take over lab populations, said Xue, who is first author on the eLife study.

“But they kept seeing that you have mixed populations at that site. So that meant it wasn’t one virus or another that was winning out; they seemed to be co-existing,” she said. And scientists at the Scripps Research Institute had made a puzzling observation: “This mutation that was coming up actually destroyed the traditional activity of the protein. So the question for us was: How could that be? That protein is really necessary,” Xue said.

When Xue and her colleagues started studying the mutant variant of H3N2 in the lab, their suspicions were confirmed.

“When we started to grow these viruses, we could barely get them to grow at all,” Xue said. “This mutation was not something that was beneficial on its own.”

Katherine Xue
UW graduate student Katherine Xue and study co-author Dr. Jesse Bloom are doing further research to see if variants of flu viruses cooperate with each other in our bodies the same way they do in cells in the lab. Photo by Robert Hood / Fred Hutch News Service

But when they combined that variant with the original H3N2 strain, the viral mixture infected cells and grew like gangbusters — certainly better together than the mutant alone and even better together than the unmutated strain alone. Working together, the viral strains were more potent, in much the same way woofers and tweeters work together to produce better sound.

The researchers also saw that when they passaged either strain through serial infections, the second variant also appeared. That is, some amount of the original H3N2 strain mutated, and some amount of the mutant H3N2 strain reverted back to the original form.

It remains to be seen whether the two variants can cooperate in a human body the way they do in cells in the lab.

To do this, Bloom and his team plan to look at viruses from the noses and lungs of patients at Seattle Cancer Care Alliance, Fred Hutch’s treatment arm. Cancer patients who come down with viral infections are followed closely and often have multiple samples taken of their resident viruses, to better understand how to treat the patients.

Xue and Bloom and their colleagues will sequence viruses taken from those patient samples, directly, without passaging them in the lab first. They’re hoping to answer questions like: “What kinds of patterns of genetic diversity can arise within a single person, within a single infection? How does that matter for the infection?” Xue said.

A theory on entrances and exits

The researchers don’t know how this specific cooperation works at a molecular level, but they have a guess.

The H3N2 variant under question harbors a single mutation in the gene coding for the protein known as neuraminidase, or NA, which sits on flu’s surface. Flu particles naturally stick to molecules on the surface of human cells, which helps them better invade those cells. Later in the virus’ life cycle, NA acts like a solvent to dissolve that molecular glue, releasing viral particles when they’re on their way out from an infected cell and freeing them to stick to their next target.

As the researchers’ theory goes, the mutant H3N2 strain may be better at entering cells because it’s stickier, missing the molecular solvent activity. But its growth could be limited alone because it has a hard time exiting cells. A mixture of the two strains could both enter and exit cells with ease — the mutant strain facilitating entry and the original strain helping with cell exit.

And because of the nature of the viral life cycle, where the host cell does all the heavy lifting of replicating and making the virus, it’s also possible that the two variants could combine in the cell. With two viral genomes to work from, the host cell might make new virus particles each carrying a mixture of the original and mutated NA protein. Two different viruses enter and one — hypothetically more potent — hybrid virus leaves.

The researchers are planning experiments to test their theory. And they also want to look for other types of flu cooperation and what kind of environments allow that cooperation to arise.

For Bloom, one of the most interesting aspects of the study was that pieces of the evidence for flu cooperation were right under researchers’ noses, in publicly available surveillance data. But nobody had thought to delve into it. So now he and Xue are wondering, in what other ways could flu cooperate to get ahead?

“The biggest value of the work is we now realize we should be thinking about that question,” he said. “As we go back and look at viral sequences that are already there, we wonder: Are similar cooperative interactions happening?”

Rachel Tompa is a former staff writer at Fred Hutchinson Cancer Center. She has a Ph.D. in molecular biology from the University of California, San Francisco and a certificate in science writing from the University of California, Santa Cruz. Follow her on Twitter @Rachel_Tompa.

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